Automatic background level compensation



AUTOMATIC BACKGROUND LEVEL COMPENSATION 5 Sheets-Sheet 1 Filed July 26,19

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3,525,369 AUIQViATlt'J BACKGRUUND LEVEL CUMPENSA'HOII Roland WernerGuhiseh, Lexington, Mass to latlonal Research Development Corporation,London, England, a corporation or Britain Filed July i963, Ser. l lo.74-7395 Claims priority, application Great Britain, .luly 31, 1967,35,130/67 Int. Cl. Gillie 21/30, 21/06; Ht'ilj 39/12 US. Cl. 2tl--2ld 6Claims ABSTRACT Uh THE DESCLUSURE stant background level. The inventionhas utility par ticularly, but not exclusively, in connection withdensitometers, that is devices that measure the variation oftransmittance or reflectance of photographic or other records in whichinformation is represented by variations in the optical properties ofthe recording medium.

In a known densitometer of the dual-beam, servo-balancing type, lightfrom a light source is split into two beams of approximately equalintensity. One beam falls upon the sample to be measured and then upon aphotomultiplier. The other beam passes through a variabledcnsity opticalfilter and then also falls upon the photomultiplier. The two beams arealternately occulted, and as long as the intensities of the beams oflight from the sample and the beam from the variable-density opticalfilter differ, the output from the photo-multiplier fluctuates. Thisfluctuating output is used to drive a motor that varies the opticaldensity of the filter in such a manner as to lessen the magnitude of thefluctuations. An equilibrium condition is finally reached in which theintensities of the two beams are equal and the optical density of thefilter is then equal to that of the sample. Should any change then occurin the optical density of the sample, the optical density of the filterwill be varied automatically until balance is once more achieved.

When such an instrument is used to examine a thin layer chromatogram,the parameter that is measured is usually the reflectance rather thanthe transmittance, but the principle remains the same, and in this casethe term optical densit is interpreted in terms of the reflectance,

a low reflectance being equivalent to a high optical density andvice-versa. 7

When a chromatogram for example is examined by such a densitometer, oneof the measurments that are made is the integral optical density of eachspot, that is, the density of the spot summed over its total area. To dothis the instantaneous optical density of any area of spot must be addedto the sum of the optical densities already recorded. Methods for doingthis are well known but their accuracy depends upon there being a veryuni form background intensity. If the background intensity of thechromatogram is not uniform then for accuracy quired to zero the counterthat registers the integral optical density of each spot, and to adjustthe densitometer continuously to compensate for variations in thebackground intensity.

it is an object of the present invention to provide upparatus foranalysing signals comprising excursions from a nominally constantbackground level, wherein there is incorporated means for automaticallyperforming such a compensating function.

According to the present invention such as an apparatus compriss meansfor producing a derivative signal representative of a differentialcoefficient of the function represented by a signal to be analysed, andmeans for establishing as a reference level, for analysis of a parameterof an excursion in this signal to be analysed, the level of the signalto be analysed at the commencement of the excursion as determined by theoccurrence of an excursion beyond a given level in the derivativesignal.

Preferably the derivative signal is representative of the seconddifferential co-cfilcient of the function represented by the signal tobe analysed, and the apparatus also includes means for detecting themaximum value of the said function during an excursion in the signal tobe analysed and the integral value of the said function over anexcursion in the signal to be analysed.

Two embodiments of the invention will be described, by way of examplewith reference to the accompanying drawings, in which,

FIG. 1 shows in diagrammatic form an automated densitometer embodyingthe present invention,

FIG. 2 shows a schematic circuit diagram of a base line compensatingcircuit according to the present invention.

FIG. 3 shows the waveforms associated with the base line compensatingcircuit of PEG. 2,

FIG. 4 shows in diagrammatic form the logic circuit shown in FIG. 1, r

FIG. 5' shows a schematic circuit diagram of a base line compensatingcircuit according to a preferred embodiment of the present invention,and,

FIG. 6 shows the waveforms associated with the base line compensatingcircuit of FIG. 5.

With relcrencc to FIG. 1, light from a source 1 falls upon an end of aflexible light guide 2 and also upon a variable-density optical filter 3through which it passes to a photo multiplier i. The flexible lightguide 2 and filter 3 are alternately occulted by a shutter 5. Theflexible light guide 2 consists of two bundles of glass fibres that aremerged together to form the shape of a capital letter Y. Although thetwo branches of the flexible light guide 2 are circular in section, theend of the main part is made rectangular in section to eilectivcly forma scanning slit 6. A chromatogram 7, or other sample to be analysed, isplaced under the slit 6 in such a manner that the wider dimension of theslit extends across the sample. The slit 6 of the light guide 2 is moveduniformly along the chromatogram 7 and just above it by a motor (notshown) until the entire chromatogram has been scanned by light from thesource 1 passing down one arm of the light guide 2. Light reflected fromthe chromatogram 7 is conveyed by the other arm of the light guide 2 tothe photo-multiplier 4. if the intensity of the light from the lightguide 2 is different to that passing through the filter 3 to thephoto-multiplier dthe output from the photo-multiplier 4 fluctuates atthe frequency of oscillation of: the shutter 5. This fluctuating outputis amplilied by an operational amplifier t; and is used to drivePatented Aun 5.9?0

a servo motor 9, which in a manner well known in the art, alters theoptical density of the filter 3 until the intensities of the two beamsof light falling upon the photo-multiplier 4 are equal and thefluctuations in output of the photo-multiplier cease. The opticaldensity of j the filter 3 is then equal to that of the chromatogram 7.The movement of the armature Flt) of the servo-motor and hence to theinstantaneous optical density of chromatogram 7. This voltage is fed toa base line compensating circuit 12, to be described more fully later,which produces a signal proportional to the instantaneous density of aspot relative to the background density of the t chromatogram 7 at theposition of the spot, and three 2 operating signals or pulses, aspot-begin pulse, a spotpeak pulse and a spot-end pulse. These signalsare treated in a logic circuit 13, also to be describedmore fully later,

and a record of the chromatogram 7 is printed by a typewriter id.

The shutter may be placed at either end of the flexible light guide 2but when it is at the end of the flexible light guide 2 nearer to thesource 1, it has the advantage of rendering the apparatus insensitive tothe effects of stray light falling upon the chromatogram 7.

When the chromatogram 7 is scanned by the flexible light guide 2, as theslit 6 comes to a spot on the chromatogram '7, a slight increase in theoptical density occurs. It is this smooth increase in density to whichthe base line compensating circuit 12 is sensitive, and which it uses toderive a reference level in relation to which the spot may be measured.This reference level is not necessarily constant, but as it is derivedat the commencement of each spot, a true measure of the increase inoptical density due to the spot may be obtained.

The base line compensating circuit is shown diagrammatically in FIG. 2,in which the triangular symbols represent operational amplifiers.

A dilferentiator 21 has applied to it at terminal T a voltage V from thepotentiometer 11, not shown, and generates a voltage V proportional tothe rate of change of V, which is applied to a circuit 22 that producesan output voltage V that is equal to the numerical value of V V isapplied to a circuit 23 which develops an output voltage V which has afirst or second value according as to whether V is above or below athreshold or reference voltage V V is applied to a sample-and-holdcircuit 24, to which V is also applied. Circuit 2:5 can either act as anamplifier of gain -1 or as a memory, so that its output voltage V; caneither follow its input voltage V but of opposite sign, or can remain ata continuous level. V and the original voltage V are applied to anadding circuit, which produces a final output voltage V at terminal Tthat is the algebraic sum of V and V Circuit 26 is identical to circuit23 and by producing a voltage V whenever V is greater than zero, whichis added to V to prevent V from falling below V during the scanning .ofa spot, operates so as to prevent confusion between the Zero value of Vthat occurs at the centre of a dark spot and that which occurs betweendark spots.

The operation of the circuit 12 is best understood with reference toFIG. 3 which shows the voltage waveforms that are produced within thebase line compensation circuit 12 as a spot is scanned by the slit 6. Asthe slit 6 begins to scan the spot the voltage V begins to rise (curves(1 and f) and so do both voltages V and V (curves b and Initially V isequal to V both in sign and magnitude. Until V exceeds the thresholdvoltage V V (curve d) remains at its first value, and circuit 24 acts asan amplifier of gain-l and V is equal and opposite to V (curve g) and Vwhich is the algebraic aszaeas sum of V and V is zero (curve It). As Vrises more steeply, so do V and V until V reaches V V then changes toits second value, producing an effective spotbegin pulse (curve e) andcausing circuit 24 to maintain V at the value it had reached when thechange in value of V occurred (curve g). As V continues to increase, Vno longer remains zero but will be the net increase in V above themaintained value of V (curve g) which represents the background level ofoptical density at the beginning of the spot. When the slit scans pastthe centre, or point of maximum density, of the spot, V drops to zeroand then rapidly increases again, but with a negative sign, as may beseen from curve 3i). V also drops to zero this point before increasingagain, but its value remains positive (curve c). This dip in the valueof V forms an effective spot-peak pulse. The input voltage to circuit 23however, does not drop to zero at the centre of the spot as it is thesum of V and V the output of circuit 26, (curve d). V 'has the formshown in curve i.

As the measured optical density represented by V drops to zero, thevoltage V reverts to its lower value, and when V also drops below itsthreshold value,V falls to its original value, thus creating a spot-endpulse. Circuit 24 then reverts to being an amplifier of gainl and V ismaintained at zero until the slit 6 begins to scan another spot.

The logic circuit 13 controls the operation of the densitometer; it isshown symbolically in FIG. 4 and operates as follows:

A switch S is closed while the chromatogram 7 is being moved intoposition. When the chromatogram 7 is correctly positioned (as determinedby a microswitch or photocell, not shown), switches 5 S and 8,; areclosed and switch S is opened. W'hen closed, switch 8; connects pulsesof fixed frequency from an oscillator 4M to a binary-coded decimalposition counter 432, the content of which is proportional to the timeelapsed from the moment when the flexible light guide 2 begins to moveand hence to the distance moved by the slit 6 from its startingposition. Switch 8;, sets all bistables and gates to their correctinitial states, and switch S causes a motor (not shown) to move slit 6uniformly across the chromatogram 7. When slit 6 arrives at thebeginning of a spot, the. spotbegin pulse from the base linecompensating circuit 12 is used to set an integral density counter 403to zero. Simultaneously, V begins to rise above zero. This .voltage isapplied to two voltage-controlled oscillators 404 and 405. Eachoscillator generates at its output a train of pulses whose frequency isexactly proportional to the input voltage; however, the oscillator 4'85is adjusted to operate at a higher frequency than the oscillator for thesame input voltage. The pulses from the oscillator 464 are applied tothe integral density counter 403 through the gate 596, and the contentof the integral density counter 303 is thus proportional to theintegrated product of optical density and time, that is, distance alongthe chromatogram.

The pulses from oscillator 495 are applied to a density counter 407through a gate 468.

As the slit 6 passes the point of maximum optical density, the spot-peakpulse from the base line coinpensating circuit 12 initiates twosequences of events.

Firstly, the contents of the density counter 407 are set to zero, thegate 408 then being opened. After a period determined by a delay 409,the gate 468 is closed and the counter 467 records the total number ofpulses that have been applied to it during the period beginning with theoccurrence of the spot-peak pulse and ending with the closing of thegate 408.

The combination of the oscillator 405, the counter 467 and the gate 498acts as a simple analogue-to-digital converter to convert the voltageproportional to the maximum density of a spot into binary-coded decimaldigits.

Secondly the spot-peak pulse causes the existing contents of the counter502 to be transferred into a print register 410 and printed by thetypewriter 1d. The bistable 411 and a gate 412 act to cause the transferto be made when none of the digits is changing.

After a time interval set by a delay 413, a gate 414- is opened and thecontents of the counter 407 is transferred to the print register 410 andprinted out, by the typewriter 14. After a further interval determinedby a delay 415, the gate 408 is opened readying it for the occurrenceof. the next spot-peak pulse. The print register 41% acts simply as abuffer storage element which is read out to the type writer l4 serially(that is, digitby-digit), and which can be loaded either in series or inparallel.

As the slit 6 passes over the end of a spot, the spotend pulse closesthe gate 4-06 so that the content of the counter 403 remains fixed.After a pause due to the delay 416, and sufficient to allow all previoustyping by the typewriter 14 to be completed, the gate 417 is opened andthe final content of the counter ill?) is transferred to the printregister ilt and the typewriter 14. After a further interval due to adelay 418 the gate 406 is opened to make the counter 403 ready for theoccurrence of the next dark spot.

When slit 6 has traversed the entire length of the chromatogram 7 switchS is closed, and switches S S and S are opened and the light guide 2 isreturned to its starting position to begin another sample.

The typewriter 14 prints a record that is in three columns: a positioncolumn in which the content of the position counter 40?. is recordedeach time it is fed into the print register did, a density column inwhich the content of the density counter 407 is similarly recorded, andan integral density column, in which the content of the integral densitycounter 403 is also recorded. This format enables the typewriter 14 toprint two or more pairs of numbers in the position and density columnswhile leaving the integral density column blank. This enablesincompletely resolved spots which have well-defincd positions and maximabut have dark areas in common, to be measured.

A preferred form of the base line compensating circuit 12 is shown inFIG. 5, in which components similar to those of the embodiment of FIG. 2have similar reference numerals.

In this embodiment the second derivative of V with respect to time V issensed, as this enables the beginning and end of a spot to be determinedwith greater precision.

This second derivative is produced by a circuit 51 that comprises two ofthe circuits 21 of the embodiment of FIG. 2 connected in series. As aresult of this modification it is possible to replace the circuit 22 ofthe embodiment of FIG. 2 by a diode 52, which merely serves to preventthe output V of 51 from becoming negative. However, this means that itis no longer possible to derive the spot peak from the signal applied tocircuit 23. The spot pealt is now taken to be when the first derivativeof V crosses zero. To observe this, a sensor 53 is connectedintermediate of the two differentiating circuits 21 of circuit 51, andis so gated by diodes 54 and 55 as to produce a spot-peak pulse at aterminal T only when a'v/dt approaches zero from a positive value.Spot-begin and spot-cur pulses are produced at terminals T and T bycircuits 56 and 57 respectively.

The remainder of the circuit functions exactly as before.

FIG. 6 shows the waveforms of the voltages that occur Within the baseline compensating circuit of FIG. 5. The waveforms that are the same asthose in FIG. 3 have the same references. As may be seen from curve jwhich shows the waveform of V the occurrence of the spot peak could beinferred from it, but the accuracy of such a method is likely to be lessthan using circuit 53 to generate a spot peak pulse an edge of whichcoincides ii with clv/dt crossing zero from a positive direction asshown in curve In. a

I claim:

1. Apparatus for analyzing signals comprising excursions from anominally constant background level, the apparatus incorporatinganalyzing means, responsive to the instantaneous magnitude of an appliedsignal, for de termining at least one parameter of each excursion in asignal to be analyzed, wherein the improvement comprises:

means for producing a derivative signal representative of a differentialcoetlicient of a function represented by the signal to be analyzed;

means for detecting the commencement of an excursion in the signal to beanalyzed by sensing the occurrence of an excursion beyond a given levelin said derivative signal; and

means, responsive to operation of the detecting means,

for applying to the analyzing means a signal which throughout eachexcursion in the signal .to be analyzed has an instantaneous magnitudeequal to the difference between (a) the instantaneous magnitude of thesignal to be analyzed and (b) the magnitude of the signal to be analyzedat the commencement of the relevant excursion. 2. Apparatus according toclaim 1 wherein the derivative signal is representative of the seconddifferential coeilicient of said function.

3. Apparatus according to claim 1 wherein said analyzing means isoperative to determine an extreme value of said function during anexcursion in the signal to be analyzed.

4. Apparatus according to claim 1 wherein said an alyzing means isoperative to determine the integral value of said function during anexcursion in the signal to be analyzed.

5. A densitometer comprising means for generating a signal related to anoptical parameter of a specimen to be analyzed, said signal comprisingexcursions from a nominally constant background level, and apparatusaccording to claim 1 for analyzing said signals.

6. In a signal analyzing apparatus including analyzing means designed todetect at least the instantaneous magnitude of signal excursions withina composite input function consisting of said signal excursions and asuperimposed nominally constant background level, an improvementproviding automatic compensation for variations in the nominallyconstant background level, said improvement comprising:

detecting means to detect the beginning of a signal excursion; and Icompensating means which provide a compensated signal to said analyzingmeans by responding to said detecting means and causing a constantcompensation value equal to the instantaneous value of said compositeinput function at the beginning of said signal excursion to besubtracted from the instantaneous value of said composite input functionduring said signal excursion,

References Cited UNITED STATES PATENTS ARCHIE R. BORCHELT, PrimaryExaminer T. N. GRIGSBY, Assistant Examiner US. Cl. X.R. 250-219;356-4201

